Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments.The objective was to develop and characterise  Bartonella bacilliformis is the causative agent of Carrion’s disease, a biphasic endemic illness of the Andean valleys. In the acute stage  severe haemolytic anaemia is present, resulting in 40–85% of deaths in untreated people and decreases to around 10% if correctly treated12Salmonella infections, among others145B. bacilliformisPeruvian wart is the chronic non life-threatening phase, characterised by cutaneous proliferative vascular lesions, occurring some weeks or months after the acute infectionAdditionally, the number of asymptomatic carriers is uncertain, although some studies have shown that 45% of the inhabitants of endemic areas present evidence of previous contact with the pathogenThe usual treatments are chloramphenicol (CHL) or ciprofloxacin (CIP), alone or combined with cephalosporins or aminoglycosides in for the acute stage. Rifampicin (RIF) or azithromycin (AZM) are used in the chronic stage6B. bacilliformis clinical isolates are scarce1113Bartonella genus11in vitro resistance to different antimicrobial agents, including coumermycin, CIP, RIF and erythromycin, has been described1718The use of antibacterial agents in the treatment of Carrion’s disease has been profuse in recent decadesin vitro obtained mutants may provide information in order to better understand antibiotic-resistance acquisition and evolution. The aim of this study was to develop and characterise a series of in vitro B. bacilliformis antibiotic resistant mutants and determine the presence of target mutations, the role of efflux pumps as well as the stability of selected resistance.Analysis of The time required for bacterial lyophilised reactivation varied from 5 weeks (strains 57.19 and 57.20) to 9 weeks (strain 57.18). Interestingly, strain 57.18 showed an initially different morphology, coinciding with the previously described T1 morphologyThe development of the antibiotic-resistant mutants required approximately 18 months, therefore, 4 antibiotic-resistant mutants were obtained from each parental strain, one for each antibiotic included in the study.Azm. Thus, after the initial 35 passages, most antibiotic-resistant mutants showed confluent growth in the presence of the antibiotic disc  except for two out of three mutants selected with CHL, which presented inhibitory halos of 18 mm (57.18Chl-35) and 32 mm (57.20Chl-35). After 5 additional serial passages , 57.20Chl-40 achieved a halo of 0 mm, while 57.18Chl-40 remained with an inhibitory halo of 18 mm . However, overall, the antibiotic requiring the least number of passages to generate confluent growth was CIP with a mean of 10.6 passages. On the other hand, CHL required >37.3 passages . Thin grof 18 mm .A maximum of 1 dilution difference between the MICs at 7 and 14 days was observed. Thus, those obtained at 7 days were used throughout the manuscript.Chl-40 mutant that reached a CHL MIC level of 4 mg/L  and >32 mg/L (CIP). The exception was the above mentioned 57.18f 4 mg/L .Azm-5St and 57.18Cip-5St. Two strains (57.18Chl-5St and 57.19Chl-5St) returned to the parental MIC levels, while the remaining isolates showed intermediate MICs between parental and final mutant values. In the case of mutants 57.19Azm-5St and 57.20Cip-5St at 14 days the presence of colonies within the inhibitory halo were observed. Thus, 57.19Azm-5St-WH showed a MIC of AZM of 32 mg/L while 57.20Cip-5St-WH showed a MIC > 32 mg/L for CIP  at L4 protein from codons 62 to 65 (Δ62–65). Nonetheless, close analysis of the spherogram showed the presence of a double sequence from the deletion onwards under the majority peaks with in the reading-frame together with a non-deleting rplD  was observed at one of the two rplD B. bacilliformis genes after position 83 . A GyrA amino acid substitution was observed in 2 out of 3 selected mutants, 57.18Cip-35 and 57.20Cip-35 , and a GyrB amino acid substitution (Glu-475 → Lys) was observed in 57.19Cip-35. The mutations present in 57.18Cip-35 and 57.19Cip-35 were stable and were also observed in the mutants 57.18Cip-5St, and 57.19Cip-5St. In the case of 57.20Cip-35 the analysis of the sequence spherogram showed the presence of a double peak with two bacterial populations, one with the WT gyrA gene sequence and other possessing the aforementioned amino acid codon substitution. This was confirmed by analysing 57.20Cip-5St (possessing a WT gyrA gene sequence) and 57.20Cip-5St-WH (which maintained the amino acid change Asp-95 → Gly). The in silico determination of the hydrophobicity pattern showed an alteration in the presence of GyrA Val-91, while the presence of GyrA Asn-95 altered both the charge and slightly affected the hydrophobicity pattern  was detected, which reverted after 5 passages without antibiotic pressure. The same mutation was also observed in the 57.20Chl-40 mutant, but a double peak was observed showing the presence of a double bacterial population, or, more probably, that the mutation was only present in one of the two 23S rRNA genes of the B. bacilliformis genome , His-540 → Tyr  and Ser-545 → Phe plus Ser-588  → Tyr (57.20Rif-35 and 57.20Rif-5St). Interestingly, when the rpoB gene in the 57.20 RIF-derived mutant at passage 13 (57.20Rif-13), was sequenced, only the presence of the amino acid codon change Ser-588  → Tyr was observed.Finally, the presence of mutations in the The study of the effect of Phe-Arg-β-Napthylamide (PAβN) and artesunate (ART) on normal bacterial growth showed that high PAβN concentrations  allowed bacterial, albeit non normal, growth. Thus, a concentration of 5 mg/L was used in the assays. No effect of ART was observed on bacterial growth with 20 mg/L. Additionally, we confirmed the lack of effect on bacterial growth with ethanol at the concentration and required volume used.Chl-40, and this effect was also confirmed by the increased disc inhibitory halo in the presence of both EPIs. Both EPIs also affected the AZM susceptibility levels of the 57.18Azm-35 mutant. Additionally, ART was found to affect the RIF MIC of 57.19Rif-35, and also enhanced the activity of RIF and CIP in 57.20Cip-35 and 57.20Rif-35 mutants respectively  to grow, support the idea that the development of antibiotic resistance has a high biological cost in B. bacilliformis, resulting in the dilution of strains carrying specific antibiotic-related mutations within a general antibiotic susceptible bacterial population. Similarly, both the loss of biological efficiency and the decrement of virulence after the development of antimicrobial resistance have been observed in other microorganisms2727The MIC of the mutants selected was higher than that of the parental isolates, achieving high MICs levels with all the antibacterial agents tested. This finding shows a worrisome scenario: the feasibility of selecting high antibiotic resistant mutants with the 4 main antibiotic families used in the treatment of Carrion’s disease. However, only those mutants selected with RIF showed consistent resistance stability, while only 1 out of 3 of CIP or AZM-resistant mutants did not revert either totally or partially. Regarding CHL, all the resistant mutants selected reverted, supporting the idea of a high biological cost of the development of CHL resistance. In the same line, the presence of double sequences when analysing some of antibiotic target gene sequences may be interpreted in three different ways: The survival of original susceptible parental strains, the partial reversion of the mutants obtained when antibiotic pressure disappears or the presence of mutations in only one of the gene copies when there is more than one. The first possibility is unlikely because of the high susceptibility levels of the parental strains to all the antibacterial agents tested  which prevent the survival of the microorganisms when the disc was placed on the center of the plate . Meanwhile the other two possibilities may be considered, and, moreover, are not mutually exclusive. They might underlie the high number of microbiological failures, isolation of viable microorganisms from blood, after successful clinical treatments23S rRNA and in the ribosomal proteins may result in concomitant resistance between CHL and macrolides3065–66 (Trp-Arg) and Δ68–69 (Lys-Gly) leading to macrolide-resistance in Streptococcus pneumoniae also confres low levels of CHL resistance. Thus, S. pneumoniae L4 Δ65–66 has been associated with an increase of the CHL MIC of up to 4-fold when cloned in a pan-susceptible S. pneumoniae strainS. pneumoniae are equivalent to positions 64 and 65 of B. bacilliformis, both included within the Δ62–65 of 57.18Azm-35 mutant. Thus, the role of L4 deletions in the acquisition of the low levels of CHL resistance observed in this mutant  is plausible, either directly due to alterations in the ribosome conformation affecting CHL binding or indirectly due to the possible induction of efflux pump overexpression as discussed below. This fact highlights the risk of co-selection of antibiotic resistance during B. bacilliformis treatment.Although non-uniform, cross effects, either increases or decreases, in the MIC levels were observed. These alterations might be explained by collateral antibiotic resistance effects that could interfere with normal bacteria biochemical processes. Three of antibacterial agents tested act at a ribosome level, while the remaining agent acts at the proteins involved in DNA decatenation and duplication. Ribosomal level alterations may differentially affect the expression of genes or may have a direct effect on the activity of other agents. In other microorganisms, specific points placed in 23S rRNA mutationsOn the other hand, selection of CHL seems to negatively affect the ability of bacteria to survive in the presence of other antibiotic agents. This fact may also be related to the above mentioned possible effect on fitness, being probably underlain by alterations in the expression or regulation of different bacterial genetic factors. Additionally, selection of hypersusceptibility to unrelated antibiotics, such as macrolides, has also been observed in relation to specific B. bacilliformis genomes. This finding, together with the high antibiotic susceptibility of the parental isolates, suggests the presence of DNA polymorphisms.Two differences leading to the substitutions Thr-13 → Ala at L4 and Arg-9 → Cys at L22, were observed between the parental and KC583 strains. Nonetheless, both substitutions were present in other 23S rRNA was observed. This latter finding differs from that described in microorganisms with a low number of 23S rRNA gene copiesB. bacilliformis and Bartonella henselae1834Regarding AZM resistance, in all the cases the presence of alterations at L4 and L22 was found, while no mutation at E. coli2636Bartonellaceae, the mutations at L4 (Gly-70 → Arg and Gly-70 → Arg + His-74 → Tyr) have been previously described in in vitro obtained macrolide-resistant mutants of B. henselaeE. coli, a series of amino acid changes involving the L4 residues Gln-62 and Gly-66 equivalent to B. bacilliformis Gln-66 and Gly-70 have been reported as being involved in the development of AZM resistance26E. coli substitutions at position Ser-70  have been describedet al.Amino acid changes at L4 and L22, including insertions and deletions, have been described in a series of unrelated microorganisms, such as B. bacilliformis. The predicted L4 deletion is located close to the L4 “hot spot” region involved in macrolide resistance in which a deletion of the 2 equivalent later amino acids in macrolide-resistant S. pneumoniae has been reportedThis is the first report describing insertions and deletions in the L4 and L22 proteins of Chl-35 and 57.20Chl-40) presented a G2372A mutation in the 23S rRNA gene. The equivalent mutation has previously been involved in CHL resistance in yeasts and E. coli25Chl-40 mutant double peaks were obtained suggesting either the presence of a double bacterial population, or, more probably, the presence of a mutation affecting only one of the two 23S rRNA genes. This fact, together with the rapid reversion of this mutation and the slow bacterial growth, which might be longer than 2 months in clinical samples cultures10E. coli results in both resistance to CHL and in retarded growth ratesTwo out of three CHL-resistant mutants  results in a decreased CIP MIC, and intermediate or low-level of nalidixic resistance4041E. coli and most other microorganisms) Ala or Val residues, as shown in The amino acid alterations observed in two out of three GyrA were located in the classical quinolone-DNA Gyrase interaction points, while resistance in the third isolate was related to the presence of mutations in the B. bacilliformis, B. henselae and Bartonella quintana181922Changes at position Asp-95, including Asp-95 → Gly, have been previously described in studies analysing quinolone-resistant mutants of B. bacilliformis. The same amino acid change at the equivalent position of S. pneumoniae (Glu-474) has been associated with quinolone resistanceS. pneumoniae a series of amino acid changes including Glu-476 → Ala and Arg-477 → His has also been described in the same region, showing the relevance of the GyrB region in the development of quinolone-resistance46Cip-5St mutant showed a MIC of 1.5 mg/L . Thus, by itself the Glu-475 → Lys alteration results in a slight increase in the CIP MIC. Moreover, this finding, together with the lack of PAβN and ART effect on the CIP MIC, highlight the presence of unstable mechanism/s of resistance in the 57.19Cip-35 mutant.This is the first description of a GyrB amino acid substitution Glu-475 → Lys in rpoB gene mutations are usually located within 3 highly conserved regions in the mid portion of the gene. In E. coli these regions include codons 505–537 (cluster I), 563–575 (cluster II) and 684–690 (cluster III)48B. bacilliformis equivalent cluster I, while Ser-588 → Tyr is located within the equivalent cluster II. Of these, the amino acid change Ser-545-Phe has been previously observed in B. bacilliformis and B. quintana in vitro RIF-resistant mutantsMycobacterium tuberculosisE. coli RpoB substitutions of Gln-513, His-526, Ser-531 or Ser-574  have been described; moreover, in some cases the same amino acid change (e.g. Ser-531 → Phe or His-526 → Tyr) has also been reported4351Rif-35) a double mutation (Ser-545 → Phe plus Ser-588 → Tyr) was observed. However, when the presence of an RpoB alteration was sought in 57.20Rif at passage 13, only the presence of the amino acid change Ser-588 → Tyr was observed. This fact shows the sequential selection of RIF resistance related to continuous antibiotic pressure. In E. coli the substitution at Ser-574  results in low-levels of RIF resistance, being its effect strongly diminished in the presence of PAβNE. coliRhodococcus equi only low levels of RIF resistance are conferredRifamycin-resistant B. bacilliformis. The two EPI tested act differently, while PAβN competes with other efflux pump substrates to be extruded from bacteria, ART diminishes the expression levels of some efflux pump encoding genes54This is the first determination of the role of efflux pumps in the development of antibiotic resistance in Azm-35 and 57.18Chl-40 mutants. In the first case the mutant presented L4 Δ62–65. Although related to deletions in L22, a similar scenario has been observed in E. coli strains carrying 3 amino acid deletions in L22 in which the inactivation of different efflux pump components results in a strong decrease in the erythromycin MIC levelsAzm-35, the use of PAβN was only visible in the disc diffusion analysis. The most probable scenario for this is that the MIC of this strain is much higher than 256 mg/L and thus, the effect on the MIC levels, albeit present, was not observed. Regarding the 57.18Chl-40 mutant both the effect of PAβN and ART decreased the CHL MIC levels to only slightly higher than the parental isolate, showing that efflux pumps may play a role in the development of low levels of CHL resistance in B. bacilliformis. No effect was observed with any inhibitor tested in either 57.19Chl-35 or 57.20Chl-40. This finding, together with the effect of ART on the antimicrobial susceptibility levels of all remaining 57.20 derivative mutants, highlight two facts: (a) the high levels of resistance to CHL derived from the presence of 23S rRNA mutations which when present mask the effect of efflux pumps, and (b) the presence of efflux pumps able to extrude the 4 antimicrobial families tested in B. bacilliformis. Thus, the different level of antimicrobial resistance of each selected mechanism may allow or not visualization of the overexpression of efflux pumps and might underlie the differences in the effect of ART on the RIF-selected mutants. ART also affects the susceptibility to RIF in 57.19Rif-35 and 57.20Rif-35 mutants, showing that efflux pumps may also affect this antibiotic in B. bacilliformis and reinforcing the possibility that the selection of a double RpoB substitution in the 57.20Rif-35 mutant was due to the low level of RIF resistance conferred. In the case of the 57.19Rif-35 mutant the effect was only observed on the MIC levels, while in the case of 57.20Rif-35 the effect was observed in the disc diameter halo. Further studies are needed to determine the mechanisms of efflux pump overexpression .Both PAβN and ART affected the susceptibility levels of 57.18B. bacilliformis to become antibiotic resistant both by the development of antibiotic-target alterations, but also mediated by efflux pump overexpression. However, regarding AZM, CHL and CIP, the instability of resistance detected in different mutants, either related to reversions and/or to dilution within a higher fitness susceptible population, suggests a high biological cost, which may underlie the rarity of antibiotic-resistant B. bacilliformis clinical isolates. Further studies analysing antibiotic target sequences from a relevant number of B. bacilliformis clinical isolates prior, during and after antibiotic treatment are necessary to better understand the natural history of antibiotic resistance in this microorganism.In summary, the present data highlight the ability of B. bacilliformis strains from the Institute Pasteur  collection were used (16S rRNA gene (Three ere used . The strRNA gene 59.B. bacilliformis on blood agar with a disc of AZM (15 μg), CIP (5 μg), CHL (30 μg) or RIF (30 μg)  which was initially put in the corner of each plate, and subsequently in the center of the plate according to halo diameterThe antibiotic resistant mutants were selected by plating the At the beginning of the study, the passages with the selected antibiotic discs were performed every other week because of the difficulty to visualise the bacterial growth in the first weeks due the large halo sizes. Then, we changed to a weekly basis, according to the evolution of each strain.Azm-35 is the AZM resistant mutant derived from strain 57.18 at passage 35).Throughout the text, the mutants obtained are referred to by indicating the name of the parental isolate, the antibiotic used in its selection and the passage number . When a mixed bacterial population was obtained , isolates from inside of halo were additionally marked as “WH” .F /MICI  was used. In all cases MIC differences >2-fold were considered as significant.The MICs of AZM, CIP, CHL and RIF of the parental isolates, the mutants at the final passage and after the 5 additional passages without antibiotic were determined by E-test . The MICs were read at 7 and 14 days. To determine the effect on the MIC levels the quotient MICIn the final passage in the presence of antibiotic discs, antibiotic cross resistance was determined by testing the MIC of each mutant with all the other antibiotics under study.B. bacilliformis were collected by adding sterile PBS into the plates and suspending the colonies with a one-use loop. This bacterial suspension was transferred to a sterile vial and the DNA was extruded by boiling at 100 °C for 10 minutes and stored at −20 °C until use.rplV and rplD genes (AZM) as well as in the 23S rRNA (AZM and CHL), quinolone resistance-determining regions (QRDR) of the gyrA, gyrB, parC and parE genes (CIP) and the rpoB gene (RIF) were determined by PCR using the primers and conditions listed in http://www.bacterio.net/bartonella.html). In cases in which the presence of non-silent mutations was observed, the deduced amino acid sequence was compared against B. bacilliformis amino acid sequences present in GenBank. Regarding GyrA, hydrophobicity was determined using the method of Kyte and Doolittle as defined in the ProtScale software60http://www.bioinformatics.nl/cgi-bin/emboss/charge).The presence of mutations in the B. bacilliformis KC583. When DNA or amino acid positions of other microorganisms are used for comparison purposes this is explicitly indicated.Throughout the text, both the DNA and amino acid numeration is referred to as that of in house with PAβN, or ART. In order to determine the EPI concentrations to be used, the effect of different concentrations of these products (from 2.5 mg/L to 20 mg/L) on normal bacterial growth was tested. Additionally, the effect of 100% ethanol (ART solvent) was also independently assessed.The role of efflux pumps in the development of antibiotic resistance was established by determining the antibiotic susceptibility levels by disc diffusion and E-test in commercial blood agar media supplemented How to cite this article: Gomes, C. et al. Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants. Sci. Rep.6, 33584; doi: 10.1038/srep33584 (2016).


It has been reported that the aqueous extract of pomegranate (AE-PG) has polyphenols with estrogenic-like activities. The present work determines if AE-PG alone or in combination with the selective serotonin reuptake inhibitor, citalopram, has antidepressant-like effects. It was also analyzed the participation of estrogen receptors (ER). AE-PG  was evaluated in ovariectomized female Wistar rats subjected to the forced swimming test. The effects induced by AE-PG were compared with those of citalopram  and 17β-estradiol . Likewise, the combination of suboptimal doses of AE-PG (0.1 mg/kg) plus citalopram (2.5 mg/kg) was evaluated. To determine if ER participates in the antidepressant-like action of pomegranate, the estrogen antagonist tamoxifen (15 mg/kg) was administered with AE-PG (1 mg/kg). AE-PG produced antidepressant-like actions with a similar behavioral profile induced by citalopram and E2. Suboptimal doses of citalopram plus AE-PG produced antidepressant-like effects. Tamoxifen was able to block AE-PG’s antidepressant-like actions. These results confirm the participation of ER in AE-PG’s antidepressant-like effects. Furthermore, the additive effects observed with the combined treatment of AE-PG plus citalopram could be advantageous in the treatment of depressive disorders, such as menopause. Punica granatum L. (Lythraceae) fruit possess several beneficial health effects for which it is considered a functional food pyrene-6-one compounds, called urolithins. These last compounds possess estrogenic activity on in vitro assays [Interestingly, the main polyphenols present in the aqueous extract of the fruit of pomegranate are ellagitannins, such as punicalagin (α and β), and flavonoids, such as anthocyanidins, catechins, and flavonols . Ellagito assays ,11. Theso assays ,13.On the other hand, the first-line treatments prescribed against menopause symptoms are antidepressants, such as selective serotonin reuptake inhibitors to treat depressive symptoms and hormone replacement therapy for hot flashes and osteoporosis ,15. HoweIn parallel, preclinical studies have reported that pomegranate reduced some menopausal signs in animal models. For instance, pomegranate seed oil consumption favors bone mineral density and prevents the trabecular damage in ovariectomized mice . AdditioNevertheless, Naveen et al. reported that the juice of pomegranate-peel extract induced antidepressant-like actions in the chronic mild stress model . These aPunica granatum (AE-PG) alone or in combination with the selective serotonin reuptake inhibitor, citalopram, produces an antidepressant-like effect and if this effect is related to estrogen receptor (ER) activation in ovariectomized rats. As reference drugs, citalopram and 17β-estradiol (E2), were tested on the forced swimming test (FST) according to previous reports [Therefore, the present work evaluates if the aqueous extract of  reports ,22,23. = 10.366; p < 0.001). In addition, the AE-PG produced an increase in swimming behavior  = 7.705; p < 0.001) without modifying the climbing behavior  = 0.428; non-significant)  = 6.039; p < 0.001) increasing the swimming behavior  = 4.478; p < 0.05), but not the climbing behavior  = 0.253; non-significant)  = 15.131; p < 0.001) and increasing swimming behavior  = 8.967; p < 0.001) while the climbing behavior was not modified by any dose  = 0.868; non-significant)  (Fificant) a. In conificant) b. Finallificant) c. = 17.841; p < 0.001) and in swimming behavior  = 12.470; p < 0.001), but not in climbing behavior  = 3.022; non-significant). = 39.908, p < 0.001 for immobility; and F = 20.217, p < 0.001 for swimming and F = 0.907, NS for climbing. The group treated with combination of the suboptimal doses of the AE-PG and the citalopram produced additive effects since there was a significant decrease in immobility   and the swimming behavior increased   when compared to the independent groups.No significant differences in locomotor activity were shown between the control group and the treated groups with the AE-PG and with E2 . HoweverThe present study determined if the aqueous extract of pomegranate (AE-PG) induced antidepressant-like effects in rats under conditions similar to those of menopause in women. To this aim, we used the forced swimming test to analyze the effects of AE-PG. This animal model of depression is one of the most common models used for evaluating pharmacological antidepressant activity ,25,26 anThe results of the present study confirm that the AE-PG induces antidepressant-like effects and that ER participate in this effect probably through an interaction with the serotonergic system. Moreover, it was determined that the combination of AE-PG plus citalopram produce an additive antidepressant-like effect.The chronic administration of the aqueous extract of pomegranate reduced immobility behavior and significantly increased swimming behavior without modifying the climbing behavior. A similar effect was induced by the typical serotonergic antidepressant citalopram. This result suggests that the pomegranate extract produces an antidepressant-like effect in ovariectomized rats. These results are consistent with two studies in male and female mice that showed an antidepressant-like effect produced by pomegranate extract under an acute treatment . HoweverBased on the behavioral profile observed in the present study, it can be suggested that AE-PG induces its antidepressant-like actions through the serotonergic system. Additionally, the behavioral effects observed were similar to those induced by the selective serotonin reuptake inhibitor citalopram and by E2. In a previous study the participation of the serotonergic system in E2 antidepressant-like effect was demonstrated ,31. TherCurrently, there are no reports in the literature showing that pomegranate extract has a direct action on the serotonergic system. A study in male mice evaluated the effects of the polyphenols obtained from the peel of the pomegranate. In this case, it was shown that pomegranate extract induces an antidepressant-like effect by inhibiting brain MAO-B and MAO-A enzyme activity by 60% and 17%, respectively . It is pIn this study, we used an aqueous extract of the whole pomegranate. According to González-Trujano et al. the polyphenols that predominate in this extract are ellagitannins  and punicallagins (5.46 ± 0.04 mg/g); and to a lesser extent, the flavonoids anthocyanidins (2.09 ± 0.23 mg/g) .Ellagic acid has been tested in the FST in mice and it has been seen that it induces an antidepressant-like effect prevented by the serotonin depletion and the antagonism to 5-HT2, 5-HT3, and α1- and α2-adrenoceptors . This daAnother possible explanation for the effect of AE-PG on swimming behavior could be given if one considers that ellagitannins can be metabolized by the microbiota to compounds called urolithins, which have activity on estrogen receptors α and β ,34. IndeThere is evidence that ERβ activation promotes changes in the 5-HT-1A receptor’s sensitivity , a recepFurthermore, it should be mentioned that current results with the chronic administration of citalopram contrast with the results previously reported . Vega-RiOur results are consistent with previous studies following sub-chronic citalopram administration, even at lower doses than those handled in the present study ,43.The combination of suboptimal doses of AE-PG and citalopram induced antidepressant-like effects; these actions were significantly higher in comparison with the individual treatments of E2 and citalopram per se. In this case, the combined treatment decreased immobility behavior by 31.9% compared to AE-PG and by 31.66% compared to citalopram. Therefore, we could say that it was observed an additive antidepressant-like effect. Concerning swimming behavior, the combination produced an increase of 62.1% compared to the AE-PG and 65.5% compared to citalopram.Generally, in clinical practice, the first choice of drug for the treatment of depressive disorders during menopause is citalopram, because its metabolism is mainly through the cytochrome p450 isoenzyme CYP2C19. Therefore, citalopram is the antidepressant that least intervenes in the metabolism of drugs prescribed for other medical complications common during menopause, such as cardiovascular illnesses .With regard to the metabolism of polyphenols, they are known to be metabolized principally through the intestinal microbiota ; the maiAn advantage of this combination is that due to the behavioral response observed and based on the literature, the inhibitory effect of the polyphenols contained in the AE-PG does not interfere with the metabolism of citalopram, so their combined use could be safe. In clinical and preclinical studies, it has been established that the combination of selective serotonin reuptake inhibitors and estrogenic compounds produce a potentiation of the antidepressant effect ,50. In pTaking into account that, in the present study, AE-PG produces its antidepressant effect through the serotoninergic system and, with the participation of ER, our results showed a pattern similar to the results obtained by Recamier-Carballo et al. In this case, an additive action of the antidepressant-like effects of a selective serotonin reuptake inhibitor (fluoxetine) occurred with E2 . Thus, tFinally, regarding the results of animals’ locomotion, in the present study, it was observed that citalopram decreased locomotion but, at the same time, in the FST, it decreases immobility by increasing swimming behavior. In this case, the effect of citalopram in locomotion is not affecting its antidepressant-like effect because a sedative effect would be seen in the FST by an increase in immobility behavior and decreased active behaviors . TherefoThis study used ovariectomized female Wistar rats (12–16 weeks old). All animals were provided by the vivarium of the “Instituto Nacional de Psiquiatría Ramón de la Fuente Muníz (INPRFM)”  and housed in groups of eight in polycarbonate cages. Animals were maintained on a 12 h inverted light-dark cycle (lights on at 10 am) in a 23–25 °C temperature-controlled room of the “Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN)” . Rats had free access to food and water. All animal procedures were performed according to the official Mexican norm (NOM-062-ZOO-1999) and approved by the Ethics Committee of the CINVESTAV-IPN No. 379-07 (23/04/2007) and INPRFM No. CEI-200 (10/12/2015).Punica granatum (AE-PG) which was kindly provided by Nutracitrus S.L. . The AE-PG was dissolved in saline solution 0.9% and administered orally ; 17-β estradiol or E2  was dissolved in corn oil and injected subcutaneously . The citalopram hydrobromide  was dissolved in saline solution 0.9% and injected intraperitoneally , and tamoxifen citrate  was dissolved in corn oil and injected subcutaneously (15 mg/kg dose). The anesthetic 2,2,2-Tribrom-ethanol 2%  was freshly prepared  and administered intraperitoneally (10 mL/kg).In this study we used a whole fruit aqueous extract of Ovariectomy was performed under 2,2,2-Tribromo-ethanol 2% (200 mg/kg) anesthesia. Ovaries were removed from the low abdominal cavity through one single midline incision which was properly sutured and treated with benzalkonium chloride 50%. Animals were kept in recovery for three weeks and then randomly divided into the experimental groups, and the behavioral studies were performed.The animals were individually placed in a glass-cylinder (45 cm height × 20 cm diameter) with water at 25 ± 1 °C and a depth of 30 cm. The FST was held in two sessions; the pre-test session, which lasted for 15 min and the test session, which lasted 5 min and which was performed fourteen days after the pre-test. There are several report that indicate that the changes induced by the pre-test remains for at least 21 days ,53,54, tThe behavioral scoring was performed to the test session by a five second sampling technique in which immobility, swimming, and climbing behaviors were recorded by an observer that was unaware of the groups’ treatments. Immobility behavior was considered when the rat did just the necessary movements to stay afloat, swimming behavior was considered when the rat moved around the cylinder or dived, and climbing behavior was considered when presenting active movements of scaling, such as moving in and out of the water with its forepaws against the glass walls .The effect of drug treatments on locomotor activity was evaluated in the open-field test just before the test session of the FST and video-recorded for later analysis. Briefly, animals were individually placed for 5 min in an acrylic cage (43 × 33 × 20 cm) with a drawn grid on the floor with 12 squares (11 × 11 cm). For the behavioral analysis, the number of times that the rat crossed the drawn squares was recorded during the 5 min session. A count was considered when the four paws were in each square. Between each session, the cage was cleaned with cleaning solution.p < 0.05 were considered as significant.Statistical analysis was performed using the Sigma Plot 12.0 Systat software . One-way ANOVA followed by a Dunnet post hoc test was used when comparing experimental groups versus the control group, and one-way ANOVA followed by a Tukey’s post hoc test when making multiple comparisons between treatments. Values of n = 7–10 rats per dose) and followed a chronic administration regimen (one dose per day during 14 days) that started twenty-four hours after the pre-test session. All behavioral tests were conducted twenty-four hours after the last pharmacological administration.For this experiment, dose-response curves were constructed as follows: (1) AE-PG ; (2) E2  as a positive control of antidepressant-like effect of estrogenic compounds; and (3) citalopram  as a positive control of antidepressant-like effects of selective serotonin reuptake inhibitors. All treatments were conducted with an independent group design  followed the same experimental design of the chronic scheme of treatment used in Experiment 1.Once the antidepressant-like effect of AE-PG was determined, the minimum effective dose of AE-PG (1 mg/kg) was evaluated in the presence of the non-selective ER antagonist; tamoxifen . For this experiment, we used four independent experimental groups of ovariectomized Wistar rats: (1) saline + corn oil; (2) tamoxifen + saline; (3) AE-PG + corn oil, and (4) tamoxifen + AE-PG. All experimental groups  followed the same experimental design of the chronic scheme of treatment used in Experiment 1.We evaluated the antidepressant-like effect of the combination of the suboptimal doses of AE-PG (0.1 mg/kg) and citalopram (2.5 mg/kg) determined from the dose-response curves constructed in Experiment 1. Additionally, four experimental groups of ovariectomized Wistar rats were used: (1) Saline; (2) AE-PG + saline; (3) citalopram + saline and (4) AE-PG + citalopram. All experimental groups (This study determined that the chronic administration of AE-PG produces an antidepressant-like effect through the modulation of the serotoninergic system and the participation of the ER. The results suggest that metabolites derived from the metabolism of ellagitannins (urolithins) may have estrogenic action. However, further studies are needed to confirm their direct involvement.Likewise, an antidepressant-like effect was observed when the pomegranate extract was combined with citalopram, suggesting an additive-type action, since we observed a summation of about 50%. According to the literature, when combining the compounds, the metabolism of one compound does not alter the metabolism of the other. However, it would be necessary to confirm that they do not have undesirable pharmacokinetic interactions.


Facilitation of linguistic experience was shown in the tonal contrast (Tone 1 vs. 3), but both age groups performed similar in the other two tonal contrasts . In Experiment 2, 6–8 and 10–12 month-old Mandarin-learning infants (n = 90) were tested with tonal contrasts that have pitch contours either similar to or inverse from lexical tones in Mandarin, and perceptual improvement was shown only in a tonal contrast with familiar pitch contours . In Experiment 3, 6–8 and 10–12 month-old English-learning infants (n = 40) were tested with Tone 1 vs. 3 contrast of Mandarin and showed an improvement in the perception of non-native lexical tones. This study reveals that tone-language learning infants develop more accurate representations of lexical tones around their first birthday, and the results of both tone and non-tone language-learning infants imply that the rate of development depends on listening experience and the acoustical salience of specific tone contrasts.To learn words in a tonal language, tone-language learners should not only develop better abilities for perceiving consonants and vowels, but also for lexical tones. The divergent trend of enhancing sensitivity to native phonetic contrasts and reduced sensitivity to non-native phonetic contrast is theoretically essential to evaluate effects of listening to an ambient language on speech perception development. The loss of sensitivity in discriminating lexical tones among non-tonal language-learning infants was apparent between 6 and 12 months of age, but only few studies examined trends of differentiating native lexical tones in infancy. The sensitivity in discriminating lexical tones among 6–8 and 10–12 month-old Mandarin-learning infants ( Infants start with a universal capacity to distinguish the phonemes of native and foreign languages , and demThere is increasing evidence to suggest that infants acquire detailed information of their native language by listening to and analyzing linguistic inputs during the first year of life . By 6 mohybrid of theories best describes the development of specific categories of phonetic discrimination , the developmental trends of lexical tones in tonal and non-tonal language learners have not been fully explored . Neverthantonese . Compareantonese . Howevers of age . These sReduced sensitivity to lexical tone contrasts among non-tonal language learners reveals that listening to an ambient language shifts the perceptual organization of lexical tones, and partially supports the attunement theory because a loss in sensitivity to tone is predicted by this model. Assessing tone perception among tonal language learners is not only necessary to reveal the developmental trends of differentiating native tone contrasts, but enhanced sensitivity to native tone contrasts is also theoretically required to evaluate attunement theory of speech perception development. In addition to listening to a tonal language, if development of tone perception depends on relative acoustical discriminability of specific tone contrasts, the perceptual learning model assumes that rate of development is slow for infants to distinguish acoustically similar tone contrasts. In other words, facilitation as well as maintenance of differentiating native tone contrasts across ages are predicted by models of speech perception.It is therefore important to assess whether the native phonological system facilitates or maintains tonal-language learning infants’ sensitivity to native tonal contrasts while non-tonal language learners change their sensitivity to non-native lexical tones. Such an investigation would help construct a better conceptual framework through which the development of native and non-native tone sensitivity could be explored between 6 and 12 months of age. Mandarin-learning infants and Cantonese-learning infants have been reported to show language-specific listening preferences for their native lexical tones at approximately 5 months of age . HoweverFigure 1 illustrates the F0 contours of the four lexical tones in Mandarin. Tone 1 is a high-level tone and Tone 4 is a high-falling tone. The pitch directions of both Tones 1 and 4 are not greatly altered within a syllable. However, Tones 2 (mid-rising tone) and 3 (low-dipping tone) exhibit similar F0 contours in isolated syllables: both have a concave F0 shape. The acoustical similarity between Tones 2 and 3 results in the frequent confusion of this tone contrast by non-tonal language speakers  distinguish four lexical tones in Mandarin; however, some tones have similar F0 contours . Figure speakers . In contspeakers . For Manspeakers . Acoustispeakers . AdoptinAlthough, both pitch height  and pitch direction (measured by the time of pitch direction change or the slope of pitch contour)  are acouwithin syllables do not change the lexical meanings of English syllables; 8- to 12- month-old English-learning infants showed an improvement in their ability to utilize prosodic patterns between syllables  in the segmentation of words and phrases from continuous speech . The acoustically most salient contrast has the largest acoustical difference, i.e., the Tone 1 vs. 3 contrast. To increase acoustical salience of tonal contrasts, the following tone contrasts were used: Tone 1 vs. Tone 3, Tone 2 vs. Tone 4, and Tone 2 vs. Tone 3. If lexical tone perception underwent a marked change between 6 and 12 months of age, the older Mandarin-learning infants would outperform the younger ones in the discrimination of native lexical tones. However, if rate of development depends on the interaction between listening experience and relative acoustical salience of tone contrasts, developmental trends of differentiating native tone contrasts would vary with tone contrasts. Improved sensitivity to discriminate tone contrasts might be observed for acoustically more salient contrasts, but maintenance of perceptual sensitivity might be shown for acoustically less salient contrasts. Experiment 2 explored whether Mandarin-learning infants relied on language-specific pitch contours to discriminate tonal contrasts, by testing the sensitivity to two tonal contrasts in which whether the tone contrasts were native to Mandarin or not was identified purely by pitch contour. The pitch contours of one tonal contrast were similar to the lexical tones in Mandarin, but contours of the other tonal contrast were inverse of the lexical tones in Mandarin. The assumption of Experiment 2 was that older Mandarin-learning infants would outperform their younger peers in discriminating tone contrasts with pitch contours similar to Mandarin tones. Experiment 3 employed a cross-language design to examine the developmental trends in the perception of non-native lexical tones among 6–8 and 10–12 month-old English-learning infants. The hypothesis was that acoustical salience of tone contrast and improvement of linguistic prosody in English-learning infants around the first birthday would also enhance English-learning infants’ ability in distinguishing tone contrasts with greater acoustical salience. In addition, if the 10–12 month-old Mandarin-learning infants demonstrated higher accuracy in discriminating Mandarin tones than the English-learning infants at the same age, it would indicate that listening to lexical tones provides additional benefits to facilitate the development of lexical tones.n = 120) participated in the study: (a) 10–12-month-olds: Tone 1 vs. 3 , Tone 2 vs. 3 , and Tone 2 vs. 4 ; (b) 6–8-month-olds: Tone 1 vs. 3 , Tone 2 vs. 3 , and Tone 2 vs. 4 . Eighteen additional infants failed to complete the testing procedures due to their inability to pass the conditioning. Results of a χ2 test on the rates of infants who could not pass the conditioning phase of the tone discrimination procedure indicated neither the age nor tone effect reached significance, at 6–8 months, χ2(2) = 0.156, p = 0.925, and at 10–12 months, χ2(2) = 0.252, p = 0.882. The pre-established criteria for inclusion in the study were that infants had no known visual or auditory deficits, were born full term (±14 days from the due date), were delivered without complications, had a normal birth weight (2.5–4.5 kg), and were developing normally. In addition, the members of the infants’ immediate families had no history of hearing loss. Parents were paid NT$ 600 for their child participating in the experiment.Two age groups of Mandarin-learning infants  language environment of Taiwanese infants was verified through a language background questionnaire, which was administrated to the caregiver before the study began. This study was carried out in accordance with the recommendations of ‘American Psychological Association ethical standards’ and ‘Research Ethics Committees of National Taiwan University’ with written informed consent from all participants. All parents gave written informed consent in accordance with the Declaration of Helsinki.hi1] (duration = 690 ms), Tone 2 [tɕhi2] (duration = 600 ms), Tone 3 [tɕhi3] (duration = 770 ms), and Tone 4 [tɕhi4] (duration = 482 ms) syllables, recorded in a sound-attenuation booth by a female Mandarin-native speaker with a normal speaking rate, and digitized with the speech analysis software, Computerized Speech Lab (CSL 4400), at a 22050 sampling rate and 16-bit resolution. The use of naturally produced speech stimuli instead of computer synthesized stimuli provided the most natural tokens by which lexical tone sensitivity in infants could be examined. Acoustical salience between tonal contrasts was reported to affect the accuracy of discriminating tonal contrasts among 1-year-old Mandarin-learning infants , 240 ms (Tone 2), 216 ms (Tone 3) and 192 ms (Tone 4), respectively. The speech samples were edited with the sound-editing software Sound Forge 7.0 . The sounds were amplified (Yamaha RX V350) and delivered to infants in an adjoining sound-treated test room via a loudspeaker (Bowers & Wilkins DM303). Parents and experimenters wore headphones (SONY MDR-CD 280) and listened to music from a CD during the tests, so they could not distinguish between the stimuli presented to the infants. Infants’ responses were monitored in the control room using a digital camera (SONY Handycam PC350) and a video monitor. Operated by an experimenter, who pushed a button on a hand-held switch, the computer used a data acquisition board  to activate the reinforcer and record the infants’ head-turn responses.The test suite consisted of two rooms. In the sound-attenuation test room, an infant was held on his or her parent’s lap, facing forward while an assistant sat at a 90-degree angle to the infant’s right side. An assistant maintained the infant’s attention by manipulating a series of engaging, silent toys to bring the infant’s gaze to midline (straight ahead of the infant). A bank of two visual reinforcers was located at a 90-degree angle to the infant’s left side, and each consisted of a dark Plexiglas box (13″ × 13″ × 13″) containing a commercially available mechanical toy . The toys were not visible until activated, at which point the lights mounted inside the box were illuminated. The visual reinforcers were placed on either side of the loudspeaker, at the infant’s eye level. A camera located in front of the infant fed an image of the test room to the adjoining control room, where an experimenter observed the infant’s behavior.hi1]), which was repeated once every 2 s, would be changed to the “target” speech sound . Pitch contour of Tones 2 and 3 are acoustically more similar than the other two lexical tones , and to reduce the possibility that large acoustical differences between target speech sounds of tonal contrasts would also contribute to the performance differences among tone contrasts, the target tone of each contrast was one of contour tones. Tone 3 was the target tone for the Tone 1 vs. 3 and the Tone 2 vs. 3 contrasts, while Tone 2 was the target tone for the Tone 2 vs. 4 contrast. The experimental protocol required a two-step training phase followed by a Test phase, all of which were computer-controlled. While the speech stimuli were playing in the background, the assistant played with toys to get the infant’s attention and distract the infant’s attention from the speech stimuli.The Head-Turn (HT) testing procedure has been previously used to explore developmental changes in consonant perception among infants 6–12 months of age . InfantsThe first step of the training phase consisted of Conditioning (+ Intensity). During this phase, infants were trained to associate the presentation of the target speech sound with the activation of visual reinforcers. The target sound interrupted the repetitive presentation of the background speech sound, and was presented at a level that was 4 dBA higher than that of the background speech sound. During the training phase, every trial was considered a target trial. The target stimulus was presented three times in a row. The onset-to-onset inter-stimulus interval was 2000 ms. The infant quickly learned to anticipate the visual reinforcer when the speech sound was changed from the background to the target. The infant had to respond to the sound change within 6 s after the first presentation of the target sound in order to watch the visual reinforcement. When the infant correctly anticipated the visual reinforcers with a head turn on two consecutive trials, the test proceeded to the next training phase, Conditioning (- Intensity).In the Conditioning (- Intensity) phase, the target sound was presented at the same intensity level as the background sound; the infants used only the phonetic difference between the sounds as a cue. All other parameters of the experiment remained the same. The infants needed to correctly produce three anticipatory head turns to advance to the Test phase. Those who failed to pass the two-phase training within 30 trials were excluded from the sample. The speech stimuli were the same in both Conditioned and Test phases, similar to those in other infant studies using the head-turn procedure . The TesIn all phases of training and testing, trials were initiated by the research assistant, who showed toys to the infants in the test room. The assistant initiated trials when infants appeared ready (focusing on the toys held by the assistant). The experimenter could not hear the stimuli presented during the trials , and was unaware of the type of trial that was automatically selected by the computer. The experimenter judged the head turn and pushed a button on a hand-held switch connected to the computer through the data acquisition board to indicate a head turn. The assistant could not hear the stimuli being presented at any time during the experiment, but was informed that a trial was underway by a small light that was automatically activated for the duration of a trial (out of the infant’s view). This was necessary information for the assistant as she was instructed not to change the toy in the midst of a trial.M = 69.86%, SD = 12.96) performed better than their 6–8-month-old counterparts , F = 51.22, p < 0.001, F = 21.55, p < 0.001, F = 18.39, p < 0.001, Figure 2 show the percentage of correct responses by infants aged 6–8 and 10–12 months while distinguishing native lexical tone contrasts.An Age × Contrast two-way ANOVA of the percentage of correct responses revealed that 10–12-month-old Mandarin-learning infants (post hoc test (p < 0.001) showed that the Tone 1 vs. 3 contrast  was easier for infants to discriminate than the other two contrasts, i.e., the Tone 2 vs. 4 contrast  and the Tone 2 vs. 3 contrast . To further examine the interaction effect between Age and Tone contrast, separate one-way ANOVAs on age effect were run for each contrast. For Tone 1 vs. 3 contrast, 10–12-month-olds  performed significantly better than their 6–8-month-old counterparts , F = 149.78, p < 0.001, p < 0.001, one-sample t-test, 6–8 month-old infants, t(19) = 8.05; 10–12 month-old infants, t(19) = 24.94. However, the perceptual improvement shown for the Tone 1 vs. 3 contrast was not observed in discrimination of the other tone contrasts. For the Tone 2 vs. 4 contrast, older infants  did not perform significantly more accurately than younger infants , F = 0.915, p = 0.345. Performance of both infant groups was significantly above chance level at p < 0.001, one-sample t-test, 6–8 month-old infants, t(19) = 6.80; 10–12 month-old infants, t(19) = 6.10. Further, for the Tone 2 vs. 3 contrast, the performance difference between the older  and younger infants  was not significant, F = 4.06, p = 0.051, p < 0.001, one-sample t-test, 6–8 month-old infants, t(19) = 7.67; 10–12 month-old infants, t(19) = 5.38. The result that Tone 1 vs. 3 contrast, the acoustically more distinct contrast, is easier than other tonal contrasts for infants to distinguish, suggests that acoustical salience between tonal contrasts affects the developmental trends of native lexical tone perception.Among tonal contrasts, the Bonferroni Results of this experiment showed that, between 6 and 12 months of age, the developmental rates of distinguishing lexical tones varied by tone contrasts. Significant improvement was observed in the Tone 1 vs. 3 contrast; this trend is consistent with previous findings that have shown an increasing sensitivity to native consonants . Howeverinverse pitch contour of Tone 3 and the non-inverse pitch contour of Tone 1.Results of Experiment 1 revealed that exposure to a lexical-tone language interacts with acoustical salience of lexical tones on the development of lexical tones perception. Pitch contour and height are acoustical cues of lexical tones, but tonal-language speaking adults perceptually weigh pitch contour more than pitch height . Would tfamiliar lexical-tone contrast, 7-month-olds  and 11-month-olds , and (2) novel lexical-tone contrast, 7-month-olds  and 11-month-olds . Thirteen additional infants failed to complete the testing procedures because of their inability to pass the conditioning phase. Results of a χ2 test on the rate of infants who could not pass the conditioning indicated neither the age nor tone contrast effect reached significance, at 7 months, χ2(1) = 0.331, p = 0.565, at 11 months, χ2(1) = 0.754, p = 0.385. The pre-established criteria for inclusion in the experiment were same as in Experiment 1. Parents were paid NT$ 600 for their child participating in the experiment. This study was carried out in accordance with the recommendations of ‘American Psychological Association ethical standards’ and ‘Research Ethics Committees of National Taiwan University’ with written informed consent from all participants. All parents gave written informed consent in accordance with the Declaration of Helsinki.The participants were 90 Mandarin-learning infants in Taiwan who were tested in two lexical-tone conditions: (1) hi], duration = 668 ms) with three patterns of pitch contour (two familiar tones and one novel tone). These lexical tones consisted of two sets of tonal contrasts in the experiment. For the familiar contrast, the pitch contours of speech stimuli were similar to Tones 1 and 3 of Mandarin. To generate the novel tone contrast, the pitch contour of one stimulus was similar to Tone 1, but the pitch contour of another stimulus was the inverse of Tone 3, and this pattern did not exist in any lexical tones of Mandarin. Figure 3 depicts the pitch contours of the speech stimuli. The pitch direction of inverse Tone 3 is generally similar to Tone 4  of Mandarin, but with the later onset of pitch falling. Therefore, combining inverse Tone 3 and non-inverse Tone 1 would generate a novel tone contrast for Mandarin-learning infants. To control the effects of acoustical salience on phonetic discrimination, the average pitch height (mean F0 = 217 Hz) and the vowel formant structures were the same for all speech stimuli, and the pitch contour was the only acoustical parameter by which to distinguish lexical tones. To generate more natural stimuli, the speech stimuli were modified from a naturally produced token using the sound-modification software, Praat  showed that older infants  performed better than younger infants , F = 6.85, p = 0.010, M = 73.47%, SD = 11.95) was easier than the novel contrast , F = 5.08, p = 0.027, F = 0.801, p = 0.373. However, given the priori hypotheses for a lack of tone contour effect at 7 months, and the contour preference emerging at 11 months, planned comparisons (simple effects tests) were conducted to verify the prediction that tone discrimination varies by pitch contour within each age group. At 7 months of age, infants performed similarly in discriminating both familiar  and novel  tone contrasts, as indicated by a planned comparison, t(42) = 0.901, p = 0.373, d = 0.274. Both 7-month-old infant groups performed above chance level at p < 0.001, one-sample t-test, familiar contour group, t(22) = 7.92; novel contour group, t(20) = 8.30. In contrast, at 11 months of age, infants were more accurate in distinguishing the familiar tone contrast  compared to the novel tone contrast , t(44) = 2.38, p = 0.022, d = 0.702. The performance of both 11-month-old infant groups was above chance level at p < 0.001, one-sample t-test, familiar contour group, t(22) = 11.94 and novel contour group, t(22) = 12.10.The results of Experiment 2 revealed that the improved accuracy in distinguishing lexical tones between 6 and 12 months of age is evident with a familiar tone contrast that contains similar pitch contours to native lexical tones, but not with a novel tone contrast whose patterns of pitch contour does not exist in the native lexical tones. Since pitch contour was the only acoustical cue for infants to distinguish lexical tones, and the performance advantage of familiar tone contrast was observed only among older infants, the results suggest that Mandarin-learning infants perceptually fine tune to the pitch contours of the lexical tones in their native language around 10–12 months of age.Results of Experiments 1 and 2 revealed that Mandarin-learning infants develop better sensitivity in discriminating the Tone 1 vs. 3 contrast around 12 months of age. However, to fully address the issue that listening to a tonal language shapes language-specific perceptions of lexical tones in early infancy, it is essential to examine whether the infants learning a non-tonal language also change their sensitivity for perceiving lexical tones. Perceptual decline in distinguishing lexical tones of a foreign language was repeatedly reported among non-tonal language learners after 9 months of age .In the present experiment, English-learning infants were tested with the Tone 1 vs. 3 contrast for which a developmental trend was clearly shown among Mandarin-learning infants in previous experiments. Therefore, the results of this experiment would be compared with those of Mandarin-learning infants in Experiment 1, and this experiment recruited only 6–8 and 10–12 month-old American infants.n = 19, mean age = 7.40 months, SD = 0.23 months, boys n = 9) and 10–12-month-old  English-learning infants. Seven additional infants failed to pass the conditioning and were excluded from the final data analysis. Results of χ2 test on the rates of infants who could not meet the criterion of conditioning phase in the tone discrimination procedure indicated neither the age nor language effect reached significance, at 6–8 months, χ2(1) = 0.168, p = 0.681, and at 10–12 months, χ2(1) = 0.138, p = 0.711. The pre-established criteria for inclusion in the study were the same as those employed in the previous experiments. Parents were paid US$ 10 for participating in this experiment. American infants were recruited through the database of names of the Infant Studies Subject Pool (ISSP) at the University of Washington. This study was carried out in accordance with the recommendations of ‘American Psychological Association ethical standards’ and ‘IRB of University of Washington’ with written informed consent from all participants. All parents gave written informed consent in accordance with the Declaration of Helsinki.This experiment included 6–8-month-old  × 2 (Infant age) ANOVA to examine the development of tone perception. Results showed that the older infants from both language backgrounds were generally more accurate than their younger peers in discriminating tone contrast, F = 56.65, p < 0.001, F = 3.32, p = 0.072. Performance of English-learning infants at both ages was above chance level at p < 0.001, one-sample t-test, 6–8 month-old group, t(18) = 3.82; 10–12 month-old group, t(20) = 10.48. However, a significant Age × Language background interaction, F = 8.60, p = 0.004, The results of the English- and Mandarin-learning infants on the discrimination of the Mandarin Tone 1 vs. 3 contrast are illustrated in M = 72.38%, SD = 9.78), who were more accurate than their younger counterparts , F = 6.45, p = 0.015, F = 0.47, p = 0.499. In contrast, at 10–12 months, Mandarin-learning infants outperformed English-learning infants in detecting lexical tone differences, F = 16.02, p < 0.001, To further examine the developmental trajectories of perceiving lexical tones in infancy, separate one-way ANOVAs were run. The results of Experiment 1 showed that the older Mandarin-learning infants discriminated the Tone 1 vs. 3 contrast more accurately than the younger infants. This perceptual improvement was also observed for the non-native lexical tones discriminated by the older English-learning infants , and pitch directions of novel tone and Tone 1 in Experiment 2 is almost parallel in the tone onset . Therefore, older Mandarin-learning infants would not perform better than younger infants in the discrimination of tone contrasts with similar onset contour. The importance of pitch onset in perceiving lexical tones was reported in Cantonese-speaking 5–6 year-old children when they identified the lexical tones with similar pitch contours  by attending to pitch contour difference, but they might extra attend to the initial segment of lexical tones  when discriminating tone contrasts with similar contours . However, older Mandarin-learning infants are not more effective than younger infants when attending to the onset rather than the whole segment of tone contour when discriminating contour tones. F0 frequency of tone onsets differ for contour tones, but the directions of pitch change in the initial part are very similar. The pitch directions of Tones 2, 3, and 4 in Experiment 1 have similar trends in tone onset (shown in contours . Future The acoustical account of tone perception development has been proposed , and sevSecond, another mechanism for developing lexical tone perception would be associated with infants’ ability to process linguistic functions of supra-segmental units, such as word stress and sentence intonation . In tonaBesides phonemic functions, lexical tones are supra-segmental units of phonetics that are expressed with speech prosody. Prosodic information of stressed syllables facilitates word segmentation for English-learning infants , and EngThe intonation of a sentence is one of the prosodic cues used to differentiate statement and question sentences. Pitch direction in certain lexical tones in Mandarin are similar to those of sentence intonations in English. The rising pitch direction of Tone 2 is similar to the intonation of questions and the falling pitch direction of Tone 4 is similar to the intonation of statements. Dutch-speaking adults were more attentive to pitch movement of Tone 2 and Tone 4 when intonations served the post-lexical function, e.g., differentiating statements and questions . In futuWould both developmental mechanisms of lexical tones compete with each other or work together for tone perception development in infancy? The present finding that 10–12-month-old Mandarin-learning infants are more accurate in detecting tonal differences of Mandarin than English-learning infants of the same age suggest that improvement in tuning to language-specific lexical tone acoustics would combine with the improving ability to perceive speech prosody for tone-language learning infants in developing their perception of lexical tones.Multiple trajectories to the development of distinguishing native lexical tone contrasts were found in Mandarin-learning infants between 6 and 12 months of age, and improving perceptual sensitivity was apparent in the Tone 1 vs. 3 contrast, the contrast with greater acoustical salience. In addition, perceptual advantage of Mandarin-learning infants utilizing familiar pitch contours was found among 8–10 month-old infants. For non-native lexical tones, older English-learning infants outperformed their younger counterparts in the discrimination of Mandarin tone contrast. In addition, 10–12-month-old Mandarin-learning infants distinguished lexical tones more accurately than English-learning infants at the same age. Therefore, this paper suggests that both the fine tuning to acoustical features of lexical tones and improving ability in processing prosodic features of supra-segmental units contribute to the development of lexical tone perception before infants’ first birthdays.F-MT conducted data collection and prepared the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Blood SOD activity in SHR was significantly higher in the AP group than in the control group. AP exerts anti-hypertensive effects on hypertension but has almost no effect on normal blood pressure. The anti-hypertensive effects of AP may be related to the anti-oxidative effects of increased blood SOD activity.We have previously demonstrated that acacia polyphenol (AP) exerts strong anti-obesity, anti-diabetic, and anti-atopic dermatitis effects. In the present study, we investigated the anti-hypertensive effects of AP. Spontaneously hypertensive rats (SHR) with hypertension and control Wistar Kyoto rats (WKY) were used. WKY and SHR were fed AP-containing food or AP-free food (control group) ad libitum for 4 weeks, and their blood pressures were measured. After AP administration, both systolic and diastolic blood pressures were significantly lower in the SHR group than in the control group. There were no differences in the systolic or diastolic blood pressure of WKY between the AP group and the control group. Angiotensin-converting enzyme (ACE) activity,  Acacia in the legume family, and it is widely distributed throughout the Australian and African continents. Acacia catechu duramen (heartwood) extract is called gambir. In Japan and China, gambir has long been used as an astringent and antibacterial agent. Polyphenol-rich aqueous extract from the bark of Acacia mearnsii De Wild. , which is widely distributed throughout South Africa, has been used to eliminate wine sediment in Europe. In addition, Australian Aborigines eat the young leaves and beans of Acacia mearnsii De Wild.Acacia is an evergreen tree belonging to the genus We have previously demonstrated that AP exerts anti-obesity and anti-diabetic actions by altering the expression of genes associated with the inhibition of obesity and diabetes in skeletal muscle, liver, and white adipose tissues . In addiIn recent years, polyphenols have been reported to have anti-hypertension effects ,5. AP coAP was administered to the WKY and SHR groups for 4 weeks, and food intake and body weight were measured. Treatment with AP did not change the food intake or body weight of WKY. There were also no changes in the food intake or body weight after treatment with AP in SHR .AP was administered to WKY and SHR for 4 weeks, and their heart weight and heart rate were measured. Administration of AP did not change the heart weight of WKY, but the heart weight of SHR decreased in a dose-dependent manner. There was a significant difference between SHR fed food with 3% AP and SHR fed normal food A. There AP was administered to WKY and SHR for 4 weeks, and their SBP and DBP were measured. The SBP and DBP of WKY fed food with 3% AP were almost the same as those in WKY fed normal food . The SBPACE is crucial in the regulation of the renin-angiotensin system; it cleaves angiotensin-I, thus yielding angiotensin-II, and hydrolyzes and inactivates the vasodilator peptide bradykinin . ACE inhAdministration of AP to WKY did not change ACE activity in the kidney. In addition, ACE activity in the cortex and medulla of the kidney did not differ between SHR receiving AP and the controls .On the basis of these results, AP did not affect ACE activity in the kidney.NADPH oxidase in the kidney was examined.Superoxide anion is a reactive oxygen species (ROS) generated by NADPH oxidase . ROS is p22 and p47 subunits of NADPH oxidase have been analyzed [p22 and p47 in the kidney of SHR  and inducible nitric oxide synthase (iNOS) in the kidney are involved in the onset of hypertension . Azuki bIn the present study, AP exhibited anti-hypertensive effects in cases of hypertension but exerted almost no effect in cases of normal blood pressure. The anti-hypertensive effects of AP might be attributed to an increase in SOD activity in the blood. Some components of AP  are absorbed by the gastrointestinal tract and exert an anti-oxidative effect in the blood. AP may have clinical applications as a functional food if the details of the mechanism of the anti-hypertensive effect of AP and its active ingredient are clarified.An RNeasy Mini Kit was purchased from Qiagen Inc. . A High Capacity cDNA Synthesis Kit was purchased from Applied Biosystems . iQ SYBR Green Supermix was purchased from Bio-Rad Laboratories . Primers were purchased from Invitrogen Corp. . A SOD Assay Kit-WST was purchased from Dojindo . All other reagents were of the highest commercially available grade.Acacia mearnsii De Wild. was pulverized and extracted for 30 min in a 10-fold volume of hot water (100 °C) and then dried using a spray drier. The extract yield was approximately 35% (w/w). The polyphenol content of the present product was 79.0%, as measured by the Stiasny reaction. AP is a complex mixture of proanthocyanidins, including unique flavan-3-ol oligomers and polymers comprising 4 or 5 monomeric units, such as robinetinidol, fisetinidol, catechin, and gallocatechin. The average molecular weight of AP is 1250 (300–3000) [AP  was donated by Mimozax Co., Ltd.  and prepared according to Cutting’s methods . Briefly00–3000) .n = 5 per group): (1) WKY-control group, (2) WKY-AP 3% group, (3) SHR-control group, (4) SHR-AP 1% group, and (5) SHR-AP 3% group. Each rat was then fed a normal diet or a normal diet containing 1%, or 3% AP ad libitum for 4 weeks. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate were monitored by tail-cuff methods  every week. After 4 weeks, blood samples were collected, and the plasma was obtained by centrifugation. The medulla and cortex were isolated from the kidney and frozen in liquid nitrogen. The animals were trained for at least 2 days prior to the BP measurements in this study. All surgery was performed under diethyl ether anesthesia, and rats were euthanized by diethyl ether inhalation.Male SHR and WKY (12 weeks old) were purchased from Japan SLC, Inc. . The rats were housed at room temperature (24 ± 1 °C) and 55 ± 5% humidity with 12 h of light . After 1 week of prefeeding, the rats were divided into the following groups (g for 90 min at 4 °C). The pellet was resuspended in buffer containing 0.5% Triton-X100. Each suspension was kept at 4 °C for 1 h and centrifuged (1000× g for 10 min at 4 °C). The resulting supernatant was used to measure ACE activity and protein content.The tissue enzyme extracts of the organs were prepared by using Masuda and colleagues’ modified method ,34. BrieO-phthalaldehyde in methanol was added. The mixture was maintained at room temperature for 10 min, and 0.1 M HCl was then added. After a 30-min incubation at 37 °C, the amount of liberated His-Leu was determined by measuring the fluorescence intensity of its adduct with O-phthalaldehyde (excitation at 390 nm and emission at 460 nm).The plasma and tissue enzyme extracts were diluted with 0.1 M HEPES buffer (pH 8.3) containing 0.3 M NaCl and 0.01% Triton-X100. The ACE reaction was initiated by the addition of the ACE substrate hippuryl-histidyl-leucine, and the mixture was incubated at 37 °C for 30 min. After the reaction was stopped by the addition of 0.1 M NaOH, 1% The levels of plasma SOD activity were measured using a SOD Assay Kit-WST, according to the manufacturer’s instructions.18S rRNA expression levels.RNA was extracted from the kidney with an RNeasy Mini Kit. A High Capacity cDNA Synthesis Kit was used to synthesize cDNA from 1 μg of RNA. Target gene expression was analyzed using real-time RT-PCR with the primers listed in p < 0.05 were considered statistically significant.Numerical data are expressed as the mean ± standard deviation. Significance was examined using Tukey’s test for multiple comparisons. Differences with a


Lolium perenne L. spp. multiflorum (Lam.) Husnot (LOLMU) is a winter annual weed, common to row crops, orchards and roadsides. Glyphosate-resistant populations of LOLMU are widespread in California. In many situations, growers have switched to paraquat or other postemergence herbicides to manage glyphosate-resistant LOLMU populations. Recently, poor control of LOLMU with paraquat was reported in a prune orchard in California where paraquat has been used several times. We hypothesize that the low efficacy observed is due to the selection of a paraquat-resistant biotype of LOLMU. Greenhouse dose-response experiments conducted with a susceptible (S) and the putative paraquat-resistant biotype (PRHC) confirmed paraquat resistance in PRHC. Herbicide absorption studies indicated that paraquat is absorbed faster in S than PRHC, although the maximum absorption estimates were similar for the two biotypes. Conversely, translocation of 14C-paraquat under light-manipulated conditions was restricted to the treated leaf of PRHC, whereas herbicide translocation out of the treated leaf was nearly 20 times greater in S. To determine whether paraquat was active within the plant cells, the photosynthetic performance was assessed after paraquat application using the parameter maximum quantum yield of photosystem II (Fv/Fm). Paraquat reaches the chloroplasts of PRHC, since there was a transitory inhibition of photosynthetic activity in PRHC leaves. However, PRHC Fv/Fm recovered to initial levels by 48 h after paraquat treatment. No paraquat metabolites were found, indicating that resistance is not due to paraquat degradation. LOLMU leaf segments were exposed to paraquat following pretreatments with inhibitors of plasma membrane- and tonoplast-localized transporter systems to selectively block paraquat intracellular movement. Subsequent evaluation of membrane integrity indicated that pre-exposure to putrescine resulted in the resistant biotype responding to paraquat similarly to S. These results strongly indicate that vacuolar sequestration is involved in the resistance to paraquat in this population of LOLMU. Lolium perenne L. spp. multiflorum (Lam.) Husnot (LOLMU) is a problem weed around the world and causes yield losses in a variety of cropping systems due to its rapid initial development, high biomass production, and plasticity  was first discovered in the mid-1950’s, and has been widely used for weed control due to its broad postemergence spectrum of weed control, non-selectivity and soil-inactivity . ParaquaFoliar absorption studies have shown that the plant cuticle is not an impediment to paraquat absorption . Uptake Arabidopsis L-type amino acid (LAT) transporter bound to the plasma membrane and an ATP-binding cassette (ABC) transporter were reported to be involved in paraquat uptake  orchard in California after several paraquat applications  and a previously characterized susceptible LOLMU (S) (Seeds from putative multiple resistant (PRHC) LOLMU plants were collected in May 2015 from a prune orchard near Hamilton City , California, 1 week after a paraquat application was made by the orchard manager. Seeds were germinated in petri dishes after the seed dormancy was overcome by alternating 5°C in darkness with 25°C in light. Seedlings were then transplanted to pots filled with Ron’s Mix soil23 stage . Plants OLMU (S)  populati-1, Gramoxone SL 2.0, Syngenta Crop Protection, LLC, Greensboro, NC, United States) at rates ranging from 105 to 6720 g a.i. ha-1, in addition to a non-treated control treatment, using a spray chamber equipped with an even flat spray nozzle and calibrated to deliver 200 L ha-1. A non-ionic surfactant  was added at a concentration of 0.25% v/v, following manufacturer’s recommendations. Pots were positioned in a completely randomized design with four replications per treatment per biotype and kept in greenhouse with daily maximum temperature of 24°C and minimum of 18°C throughout the experiment. Visual injury was evaluated 7, 14, 21, and 28 days after treatment (DAT) using a scale 0–100%, where 0% represents absence of visual injury and 100% represents complete mortality. At 28 DAT, above ground biomass was collected, dried, and weighed. Log-logistic regression was used to obtain growth reduction by 50% for both biotypes (GR50) and the resistance index (RI)  . The exp14C-paraquat . Radiolabeled herbicide was mixed with a solution containing commercial paraquat (Gramoxone 2.0 SL) and non-ionic surfactant , to yield a final concentration approximating a spray solution of 105 g a.i. ha-1 and 0.25% v/v, respectively. A 1-μL droplet of the solution was placed on the adaxial leaf surface of the youngest fully expanded leaf, 2 cm away from the leaf ligule towards the leaf blade apex, using a blunt-edged syringe (-2 s-1 PAR) for an additional 14-h period  and 14C-carbon disintegration measured with a liquid scintillation spectrophotometer . Treated leaves, shoot and roots were oven-dried and then combusted in a sample oxidizer  where 14CO2 was trapped in a specific CO2 trapping solution , mixed with the appropriate scintillation cocktail , and 14C decay quantified with liquid scintillation techniques. Each treatment by harvest combination was replicated four times and the experiment was conducted twice. Data were pooled following the same criteria as the whole-plant dose-response experiment. Absorption of 14C-paraquat was calculated as percentage of applied and translocation as percentage of absorbed. Absorption data were subjected to non-linear regressions . Falcon tubes were sonicated for 30 min at 65°C, centrifuged at 3800 g for 45 min, and a 1-mL aliquot was collected from the supernatant phase. To eliminate particulate matter in the 1-mL aliquot, samples were filtered with a 0.45 μm PVDF syringe filter  prior to being transferred to 2-mL injection vials. An HPLC  equipped with a mixed-mode column  in line with a flow-through radioactivity detector  was used to quantify the parent compound and observe any potential metabolites. The mobile phase was composed of 25% ammonium acetate  and 75% acetonitrile , the column oven temperature was set to 30°C and flow rate 0.6 ml min-1. The experiment was conducted using a completely randomized design with four replications per biotype at each time point and the experiment was repeated.PRHC and S were grown, dark- and light-incubated, and treated as described in the absorption and translocation section. In this experiment, 16.6 kBq of -1. Plants were kept in a growth chamber set at 24°C, 14/10 h day/night, and 800 μmol m-2 s-1 PAR. To assess the plant photosynthetic performance after exposure to paraquat, the maximum quantum yield of photosystem II (Fv/Fm) was measured by dark-adapting the marked leaves with dark-adaption clips  for 20 min prior to taking chlorophyll a fluorescence measurements with a chlorophyll fluorometer  , along with 0.25% v/v non-ionic surfactant (Activator 90), was applied at 105, 420, 840, and 3360 g a.i. ha States) . Fv/Fm mN-morpholino)ethanesulfonic acid pH 6.5  and 0.1% Triton X-100.An electrolyte leakage technique  was adop-2 s-1 PAR was applied for 12 h to allow paraquat action.After 3 h of incubation in the inhibitor solutions, leaf segments were rinsed and transferred to glass scintillation vials containing 5 mL of solution [2% sucrose (w/w) + 1 mM MES] with or without paraquat [25 μM paraquat] and incubated for 14 h. Vials were arranged in a completely randomized design in a growth chamber set at 24°C throughout the experiment. Treatments containing paraquat were used to assess the role of the transporters in the resistance phenotype and treatments without the herbicide were to correct for background effects. After dark incubation, 800 μmol m-2 s-1 PAR for 24 h followed by four freeze-thaw cycles . The experiment was repeated and data were pooled using criteria previously stated. Data were analyzed as a 6 by 6 factorial, with treatments as the main factors and incubation time as the subfactor.Conductivity measurements were carried out with a conductivity meter and probe . An initial measurement was taken when leaf segments were transferred to solutions (0 HAT) to use as background conductivity. Measurements were also taken at 11 (dark), 14 (dark), 19 (light), 22 (light) and 26 HAT (light). Each measurement was standardized as a percentage of the maximum conductivity of the sample, obtained by exposing samples to 2000 μmol m-1 (data not shown). Lower paraquat rates (105 and 210 g a.i. ha-1) did not reduce PRHC biomass, whereas these rates reduced S biomass by more than 50% compared to the non-treated control . Half of the recommended field rate (240 g a.i. ha-1) reduced the biomass of S to near 0%, whereas eight times the recommended rate (6720 g a.i. ha-1) was required to induce a comparable response in PRHC. Three-parameter log-logistic regressions were the best fit for the dose-response data. Large standard errors were observed when S data were modeled, particularly the estimated GR50, presumably due to the high susceptibility of S to paraquat even at low herbicide rates (Table 1). The regression estimate e (GR50) was 59 g a.i. ha-1 for S and 1780 g a.i. ha-1 for PRHC, resulting in a 30-fold RI.Paraquat damage was observed in S at all herbicide rates as early as the first visual assessment (7 HAT). Conversely, damage to PRHC leaves was only visible at rates higher than 210 g a.i. ha14C-paraquat into the treated leaf over time reached the upper limit by 16 HAT, within the dark-incubation period, and was best described by rectangular hyperbole models . Based on the regression estimate 𝜃 = 90t (time required to 90% of the maximum absorption to be achieved), absorption into S treated leaves was faster (P < 0.001) compared to PRHC (Table 2). Conversely, maxA, which represents the maximum absorption percentage of the 14C-paraquat applied, was similar for both biotypes.Absorption of 14C-paraquat, the herbicide movement out of the treated leaf is limited in both biotypes (data not shown). For this reason, light conditions before and during the absorption and translocation experiment were manipulated to allow 14C-paraquat to translocate before its activity resulted in tissue damage. Translocation of 14C-paraquat out of S leaves increased in an exponential fashion , reaching 56% by 30 HAT , whereas movement out of PRHC leaves exhibited a linear response and less than 3% of 14C-paraquat was detected in plant parts other than the treated leaves. Paraquat exudation into the hydroponic solution was negligible (data not shown). The methodology adopted to evaluate the absorption and translocation of 14C-paraquat yielded total recovery of 97.3 ± 2.9% .Preliminary studies indicated that, when plants are light-incubated after treated with 14C-paraquat. A linear response was obtained with the in-line radioactivity detector (R2 = 0.99) over the range of 14C-paraquat concentrations of 0.8 to 33.3 Bq μL-1, with limits of detection lower than 0.8 Bq μL-1 . Elution of 14C-paraquat occurred at 2.37 min after sample injection, and no other 14C peaks were observed in samples from PRHC and S (data not shown) suggesting a lack of paraquat metabolism.The extraction procedure recovered >98% of the applied v/Fm values as a consequence of oxidative damage and loss of photosystem II reaction centers. The lowest rate of paraquat (105 g a.i. ha-1) did not reduce PRHC Fv/Fm, whereas Fv/Fm in S plants was reduced to less than 10% of the non-treated control up to 48 HAT . Half (420 g a.i. ha-1) and full (840 g a.i. ha-1) of the recommended paraquat field rates transiently reduced PRHC Fv/Fm up to 5 HAT, but the photosynthetic performance recovered by 48 HAT, whereas Fv/Fm values in S plants dropped to zero by 48 HAT. The highest rate of paraquat (3360 g a.i. ha-1) reduced Fv/Fm in both biotypes to 0% compared to the initial values.Plants exposed to biotic and abiotic stresses exhibit decreases in FFigure 5). PRHC leaf segments treated with only paraquat exhibited the lowest electrolyte leakage on average; values were statistically similar to leaf segments pre-treated with verapamil. Sodium-orthovanadate and potassium nitrate increased susceptibility of PRHC leaf segments to paraquat in comparison with paraquat-only treatments. Lastly, pre-treatment with putrescine, a polyamine transport inhibitor, followed by paraquat increased electrolyte leakage of PRHC leaf segments to levels similar to S leaf segments treated with paraquat-only, essentially reversing resistance to paraquat.The technique employed to assess the behavior of paraquat in cells of LOLMU pre-treated with inhibitors produced consistent and reproducible results . Non-target-site mechanisms of resistance are extensively reported in the literature, and particular attention to these types of mechanisms is given when paraquat-resistant biotypes are studied  are translocated in plants by long-distance transport systems  transiently reduced Fv/Fm, in PRHC but the photosynthetic apparatus recovered by 48 HAT, suggesting not only that paraquat reaches PRHC chloroplasts, but also that the mechanism of resistance to paraquat does not involve herbicide exclusion from the plant cell as suggested for Hordeum leporinum , the time in which paraquat is in the cytoplasm is similar in PRHC and S, potentially with a paraquat exclusion mechanism in the chloroplasts preventing paraquat from reaching its site of action. This chloroplast exclusion mechanism might not be sufficiently expressed to eliminate damage from high paraquat doses but may be sufficient at lower doses, explaining the results obtained with the maximum quantum yield of PSII measurements. Reduction of paraquat accumulation in Arabidopsis chloroplasts with the gene PAR1, which encodes a Golgi-localized LAT transporter, has been shown to confer tolerance to the herbicide  maintaining the linear fashion of the natural vacuolar loading observed in Z. mays (Studies with the sub-cellular compartmentation of paraquat in paraquat-susceptible aturable . If it ierbicide . However Z. mays .2+ channels (2+ channels and multidrug ABC transporters are not involved in the resistance to paraquat in this biotype. The inhibitor of plasma-membrane, tonoplast ATPases and plasma-membrane ABC transporters sodium-orthovanadate (+-ATPase pumps inhibitor potassium nitrate (Verapamil, which blocks Cachannels  and inhichannels  did not vanadate , as well nitrate , led to  nitrate .Z. mays roots to putrescine 20 min prior to paraquat incubation has been shown to inhibit polyamine transporter-mediated paraquat transport by up to 65% (The observation that pre-exposure of PRHC leaf segments to putrescine reverses resistance to paraquat strongly suggests that polyamine carrier systems are involved in the mechanism of resistance to paraquat in PRHC. Exposure of p to 65% .Polyamine transport can be accomplished by carriers bound to the plasma membrane  and tono14C-paraquat in PRHC observed seems to be primarily caused by the vacuolar sequestration of the herbicide mediated by tonoplast-bound polyamines transporters sensitive to the inhibitor putrescine. These findings are supported by the fact that PRHC photosynthetic apparatus is sensitive to paraquat and that paraquat is stable in the plants.Poor weed management practices, particularly overreliance on a single/few herbicide modes of action, have frequently been associated with the selection of herbicide-resistant weed biotypes around the world. Paraquat used to control glyphosate-resistant LOLMU has selected for a multiple resistant biotype in a prune orchard in California; this biotype withstands paraquat at up to three times the maximum field rate that tree and vine growers are allowed to use in this region. The restricted translocation of CB and BH conceived and designed the experiments. CB conducted the experiments and analyzed the data. CB and BH wrote the paper. All authors have read and approved this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.